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Norovirus GI, GII and GIV nucleic acid genotyping reagent kit and detection method

A detection kit and virus nucleic acid technology, applied in the field of GII and GIV type nucleic acid typing detection kits, and norovirus GⅠ, can solve the problem that norovirus detection methods have not been reported, and achieve the promotion of primer annealing and unique design. , the effect of reducing workload

Active Publication Date: 2019-09-24
伯杰(青岛)医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Domestic fluorescent quantitative PCR detection reagents for norovirus are mainly based on simple detection of norovirus GI and GII types. Chinese patent document CN101153341A discloses primers, detection methods, and detection kits for norovirus GII type detection. Chinese patent document CN108085414A Norovirus GI and GII detection primers and probe sets have been disclosed, but there are no reports on the detection methods for the three pathogenic norovirus GI, GII and GIV types

Method used

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  • Norovirus GI, GII and GIV nucleic acid genotyping reagent kit and detection method
  • Norovirus GI, GII and GIV nucleic acid genotyping reagent kit and detection method
  • Norovirus GI, GII and GIV nucleic acid genotyping reagent kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1——Norovirus GⅠ, GⅡ, GⅣ nucleic acid typing detection kit

[0046] A kit for multiple detection of Norovirus GⅠ, GII and GIV types by real-time fluorescent RT-PCR, including: virus nucleic acid rapid extraction reagents, PCR amplification reagents, PCR enzyme mixture, Norovirus GⅠ, GⅡ and GIV types Nucleic acid detection reagents, positive control substances, negative control substances. in:

[0047] (1) The viral nucleic acid rapid extraction reagent includes a lysis reagent, and the lysis reagent comprises: 100mM Tris-HCl with a pH of 10.0, 5% v / v Tween20, 4M guanidine isothiocyanate, 50mM KCl, and 2mM EDTA with a pH of 8.0.

[0048] (2) The PCR amplification reagent includes PCR amplification buffer; specifically includes: 2×PCR buffer, 50mM MgCl 2 , 100 mM Tris-HCl, pH 10.0, 0.5 mM dNTPs, 5% g / mL BSA.

[0049] (3) The PCR enzyme mixture includes 0.5 U / μl reverse transcriptase, 5 U / μl DNA polymerase, and 5×RNase inhibitor.

[0050] (4) The Norovirus GI, G...

Embodiment 2

[0053] Example 2——Norovirus GⅠ, GⅡ and GⅣ type nucleic acid typing detection method

[0054] A real-time fluorescent RT-PCR multiple typing detection method for Norovirus GⅠ, GII and GIV types, comprising the following experimental steps:

[0055] (1) Main reagents and instruments: the kit reagents in Example 1 were used; the fluorescent quantitative PCR instrument was ABI7500.

[0056] (2) Specimen preparation: the positive samples were norovirus GⅠ positive patient samples, norovirus GⅡ positive patient samples, norovirus GIV positive patient samples, and then diluted with DEPC water in different multiples, negative control The specimens were samples from patients negative for norovirus GⅠ, GⅡ and GⅣ infection.

[0057] (3) Nucleic acid extraction: Mix equal volumes of the viral nucleic acid rapid extraction reagent and the sample to be tested, let stand at room temperature for 5-10 minutes, and then directly use the lysed mixture for PCR detection.

[0058] (4) Specific R...

Embodiment 3

[0094] Example 3——verify the influence of KCl in the rapid extraction reagent of viral nucleic acid on the detection effect of fluorescent PCR

[0095] (1) According to the formula provided by the present invention, the viral nucleic acid rapid extraction reagent A containing KCl is prepared: pH is 10.0 100mM Tris-HCl, 5%v / v Tween20, 4M guanidine isothiocyanate, 50mM KCl, pH 8.0 2 mM EDTA.

[0096] (2) Prepare KCl-free viral nucleic acid rapid extraction reagent B: 100 mM Tris-HCl with a pH of 10.0, 5% v / v Tween20, 4M guanidine isothiocyanate, and 2 mM EDTA with a pH of 8.0.

[0097] (3) Using norovirus GⅠ, GⅡ and GⅣ positive clinical samples, mix the virus nucleic acid rapid extraction reagent A and viral nucleic acid rapid extraction reagent B with equal volumes of the sample respectively, let stand at room temperature for 5-10 minutes, and then lyse and mix Solutions A and B were used for PCR detection respectively.

[0098] (4) The norovirus GⅠ, GII and GIV fluorescent P...

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Abstract

The invention discloses a Norovirus GI, GII and GIV nucleic acid genotyping reagent kit. The reagent kit comprises a viral nucleic acid quick extraction reagent, a PCR amplification reagent, PCR enzyme mixed liquor, a Norovirus GI, GII and GIV nucleic acid detection reagent, a positive control product, and a negative control product. The formula can be used for quick extraction of virus RNS, and the PCR detection efficiency is improved. The Norovirus GI, GII and GIV nucleic acid detection reagent comprises a Norovirus GI amplification primer F1, R1 and probe P1, a Norovirus GII amplification primer F2, R2 and probe P2 and a Norovirus GIV amplification primer F3, R3 and probe P3; and the sequence of the primer probe is created for the first time and unique in design, good in specificity and high in sensitivity. The invention further provides a Norovirus GI, GII and GIV nucleic acid genotyping detection method. According to the Norovirus GI, GII and GIV nucleic acid genotyping reagent kit and detection method disclosed by the invention, whether Norovirus GI, GII and GIV pathogens are contained in samples to be detected can be detected in a multichannel manner at the same time, quick and precise detection can be realized, viruses can be quantified, and application is convenient.

Description

technical field [0001] The invention belongs to the technical field of in vitro diagnostic reagents, and relates to an RT-PCR amplification technology, in particular to a norovirus GⅠ, GII and GIV type nucleic acid typing detection kit and detection method. Background technique [0002] Norovirus, also known as Norwalk Viruses (Norwalk Viruses, NV), is a virus belonging to the Norovirus (Norovirus, NV) genus in the Human Calicivirus (HuCV) family. Slightly different virus particles. Norovirus infectious diarrhea is prevalent all over the world, and infection can occur throughout the year. The infected objects are mainly adults and school-age children, and the incidence is high in cold seasons. Noroviruses are divided into 5 genomes (GⅠ-GⅤ), of which only GⅠ, GⅡ and GⅣ can infect humans, and GⅢ and GⅤ infect cattle and mice respectively. Norovirus has strong resistance to the external environment, low infection dose, short incubation period after infection, long detoxificat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851
CPCC12Q1/6851C12Q1/701C12Q2600/16C12Q2600/166C12Q2531/113C12Q2563/107C12Q2537/143C12Q2545/113
Inventor 蒋小琴朱兆奎赵百慧
Owner 伯杰(青岛)医疗科技有限公司
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